Improving tobacco agronomic and chemical properties is a complex goal for breeders, as development of genetic markers and mapping strategy are hampered by the lack of polymorphism within Nicotiana tabacum . A starting point for discovering Quantitative Trait Loci (QTLs) linked to leaf and smoke properties is presented. A Recombinant Inbred Line population was developed from an intraspecific cross between two flue-cured Nicotiana tabacum L. inbred lines with unrelated breeding origins. A total of 59 traits, related to diverse agronomic, leaf quality, chemical composition and smoke properties were assessed. Near Infrared Reflectance Spectroscopy (NIRS) analyses of leaf lamina powders and mechanical smoking under a standard ISO regime were used to estimate chemical traits and smoke mutagenicity of the main smoke stream. The 114 RILs were screened for 184 AFLP (Amplified Fragment Length Polymorphism), ISSR (Inter Simple Sequence Repeat), SSAP (Sequence Specific Amplified Polymorphism), and SCAR (Sequence Characterized Amplified Region) markers. Loci involved in resistance to PVY^N (Potato Virus Y necrotic strain) and black root rot ( Chalara elegans ) were also mapped. Out of all tested markers, 75% were mapped on a partial genetic map including 18 linkage groups. Substantial segregation distortion (47%) was observed in linkage groups throughout the genome. A total of 75 significant QTLs were detected for 52 traits by simple and composite interval mapping, with 8 to 41.5% of the variation in the examined traits. Nineteen QTLs had opposite effects to those expected from the ranking of parental means.